Project Abstract Cellular differentiation is a fundamentally important process, whereby highly-diverse cellular phenotypes are derived from a single underlying genome through the differential regulation of gene expression across time and space. Epigenetic information, non-genetic heritable changes in gene regulation, is integral to proper cellular differentiation and organismal development, with many developmental disorders characterized by errors in epigenetic regulation. Fundamental to both cellular differentiation as well as organismal plasticity is the appropriate epigenetic regulation and demarcation of cell- or phenotype-specific enhancer elements. Despite this, our current understanding of how enhancers are epigenetically poised, activated, and remembered during differentiation is still quite limited. Without a better understanding of the differential epigenetic regulation of enhancers, our ability to treat complex developmental disorders such as Rett syndrome and Autism, as well as fully understand plasticity-associated pathologies such as obesity will remain limited. The eusocial insect Harpegnathos saltator provides an excellent, experimentally-tractable system within which to study the epigenetics of differentiation and dynamic plasticity, as H. saltator workers possess the ability to transition between physiologically-distinct reproductively inactive and active (gamergate) states, based entirely upon environmental cues. Our preliminary data indicate that this reproductive plasticity seen in H. saltator is underlain by preferential epigenetic activation of worker- and gamergate-specific enhancers (often bound by CBP). Furthermore, we observe that enhancers active only in gamergates show preferential enrichment for H4K16 acetylation in workers. Importantly, emerging evidence in vertebrate systems has implicated H4K16ac and its associated acetyltransferase MOF, in the regulation of key developmental enhancers and maintenance of pluripotency. The goal of this proposal is to use the experimentally reversible reproductive plasticity seen in H. saltator workers to test the hypothesis that H4K16ac is integral for the marking of plasticity-associated eukaryotic enhancers, poised for activation upon differentiation. We will assess our hypothesis by evaluating the extent to which H4K16ac and its mediator (MOF) establish and maintain poised enhancers for reproductive plasticity in ants (Aim 1), as well as test how observed age- dependent decreases in worker reproductive plasticity relate to changes in H4K16ac and other histone PTMs at these enhancers (Aim 2). For both aims we will use the inducible reproductive plasticity of H. saltator workers as a model to study the epigenetics of differentiation. We will also utilize targeted manipulation of histone PTMs (using drugs and RNAi) to evaluate the causality of our findings, and comparative techniques to assess their generality. These experiments will greatly inform our view of enhancer activation as it relates to differentiation, as well as deepen our understanding of the epigenetic regulation of metazoan plasticity.